Enhanced myogenic differentiation by extracellular matrix is regulated at the early stages of myogenesis

Myogenic cell lines have been used extensively in the study of skeletal muscle development, regeneration, and homeostasis. To induce myogenic differentiation, culture media composed of a wide variety of growth factors and other additives have been used. Because the diversity in these components may modulate the differentiation process differentially, we describe a differentiation protocol that does not require the introduction of any factors to the differentiation media (DM) other than those present in the growth media. By culturing C2C12 skeletal myocytes on a coating of diluted Matrigel, a soluble basement membrane, consisting of collagen IV, laminin, heparan sulfate proteoglycans, and entactin, myogenic differentiation was accomplished by mere serum reduction. Assessment of myotube formation, creatine kinase activity, myosin heavy chain-fast, and myogenin demonstrated that the kinetics and extent of myogenic differentiation were superior using this protocol, compared with a commonly used differentiation protocol, in which an extracellular matrix is not provided and the DM contains horse serum. In addition, the elevated transactivation of a troponin-I promoter reporter construct suggested that myogenesis was enhanced at the transcriptional level. Finally, assessment of genomic deoxyribonucleic acid content revealed that the Matrigel differentiation protocol resulted in lowered proliferation. This protocol may aid studies aimed at elucidating mechanisms of myogenic differentiation, where a homogeneous population of myotubes is preferred.